The effects of interleukin-1 on the expression of thrombospondin and fibronectin by rabbit articular chondrocytes

Studies to evaluate the effects of recombinant interleukin-1 beta (IL-1) on the expression of matrix proteins by rabbit articular chondrocytes were conducted. Chondrocytes expressed high levels of message for thrombospondin (Tsp) and fibronectin (Fn). RNA slot-blot analysis demonstrated that treatment of the cultures with IL-1 (100 ng/ml) for 24 h caused a 70% suppression of their steady-state Tsp mRNA levels whereas those of Fn were not affected. Steady-state mRNA levels for the intracellular protein, actin, were not modulated by treatment with IL-1. The suppression of Tsp mRNA levels by IL-1 (100 ng/ml) was maximal by 4 h and was concentration dependent; half-maximal suppression was estimated to require 0.12 ng/ml IL-1. Cycloheximide treatment enhanced Tsp mRNA levels, but did not modulate IL-1 suppression of Tsp mRNA. Using pulse-labeling and immunoprecipitation techniques, we found that IL-1 suppression of Tsp mRNA levels was reflected in a coordinate inhibition of Tsp protein synthesis. Chondrocyte synthesis of Fn was not affected by IL-1. These data suggest that IL-1 specifically regulates chondrocyte expression of Tsp at least in part by decreasing the amount of Tsp mRNA available for translation.     

Modulation of H+ transport mechanisms by interleukin-1 in isolated bovine articular chondrocytes.

The proinflammatory cytokine interleukin-1 (IL-1) promotes the degradation of articular cartilage by inhibiting matrix synthesis and stimulating degradative enzyme activity. Generation of nitric oxide (NO) in response to IL-1 is implicated in these actions. The catabolic actions of IL-1 can be inhibited by manoeuvres which are predicted to dissipate H+ gradients across the chondrocyte plasma membrane. In the present study, the effects of IL-1 on H+ extrusion from bovine articular chondrocytes were investigated. pH was measured using the H+-sensitive fluorescent dye BCECF. Cells were acidified by ammonium rebound and the contribution of the Na+-H+ exchanger (NHE) and of the vacuolar H+-ATPase to acid extrusion was characterised by ion substitution and inhibitor studies. Overnight (18 h) exposure to IL-1 stimulated acid extrusion in a dose-dependent fashion. This effect represented stimulation of both NHE and the ATPase. Characterisation of the timecourse of this response indicated that, while stimulation of acid extrusion was rapid, effects on the ATPase were only apparent after greater than 8h incubation with the cytokine. In keeping with this observation, the protein synthesis inhibitor cycloheximide abolished the stimulatory effect of IL-1 on ATPase-mediated extrusion. The upregulation of ATPase activity by IL-1 was inhibited by the NOS inhibitor L-NAME and by the NO scavenger PTIO. In cells which had not been exposed to IL-1, treatment with the NO donor SNAP also stimulated acid extrusion by the ATPase. In contrast, NHE activity was not altered by any of these compounds. Taken together, these results imply that IL-1 can stimulate acid extrusion in chondrocytes and that this reflects rapid upregulation of NHE with slower induction of H+-ATPase activity which requires elevated levels of NO. While ATPase induction involves protein synthesis, this process may not constitute synthesis of ATPase proteins per se, but rather of some associated regulatory process.     

Gene expression profiling of embryonic human neural stem cells and dopaminergic neurons from adult human substantia nigra

Neural stem cells (NSC) with self-renewal and multipotent properties serve as an ideal cell source for transplantation to treat neurodegenerative insults such as Parkinson's disease. We used Agilent's and Illumina Whole Human Genome Oligonucleotide Microarray to compare the genomic profiles of human embryonic NSC at a single time point in culture, and a multicellular tissue from postmortem adult substantia nigra (SN) which are rich in dopaminergic (DA) neurons. We identified 13525 up-regulated genes in both cell types of which 3737 (27.6%) genes were up-regulated in the hENSC, 4116 (30.4%) genes were up-regulated in the human substantia nigra dopaminergic cells, and 5672 (41.93%) were significantly up-regulated in both cell population. Careful analysis of the data that emerged using DAVID has permitted us to distinguish several genes and pathways that are involved in dopaminergic (DA) differentiation, and to identify the crucial signaling pathways that direct the process of differentiation. The set of genes expressed more highly at hENSC is enriched in molecules known or predicted to be involved in the M phase of the mitotic cell cycle. On the other hand, the genes enriched in SN cells include a different set of functional categories, namely synaptic transmission, central nervous system development, structural constituents of the myelin sheath, the internode region of axons, myelination, cell projection, cell somata, ion transport, and the voltage-gated ion channel complex. Our results were also compared with data from various databases, and between different types of arrays, Agilent versus Illumina. This approach has allowed us to confirm the consistency of our obtained results for a large number of genes that delineate the phenotypical differences of embryonic NSCs, and SN cells.     

Recombinant human interleukin-1 inhibits plasminogen activator inhibitor-1 (PAI-1) production by human articular cartilage and chondrocytes

Human articular cartilage and chondrocyte monolayers in culture constitutively produced plasminogen activator inhibitor-1 (PAI-1) protein and mRNA, as assessed by a specific enzyme-linked immunosorbent assay and Northern blotting analysis, respectively. Recombinant human interleukin-1 (IL-1) invoked a dose-dependent inhibition of PAI-1 production in both cartilage and chondrocyte cultures. The inhibitory effect of IL-1 was observed between 2-8h after addition of the cytokine, while the optimal dose was between 10-100U/ml IL-1 alpha (57-570pM IL-1 alpha). Results obtained by Northern analysis of chondrocyte total RNA reflected those found for the PAI-1 antigen, namely, that nontreated chondrocytes showed PAI-1 mRNA which was reduced by IL-1 treatment. To our knowledge, this is the first report where IL-1 has been found to inhibit PAI-1 expression. Since IL-1 has been shown before to cause human cartilage destruction and a correlated change in plasminogen activator activity, it could be that a concomitant reduction in PAI-1 levels by IL-1 may be significant in the control of these changes in cartilage.     

Gene expression profiling of human diseases by serial analysis of gene expression

Until recently, the approach to understanding the molecular basis of complex syndromes such as cancer, coronary artery disease, and diabetes was to study the behavior of individual genes. However, it is generally recognized that expression of a number of genes is coordinated both spatially and temporally and that this coordination changes during the development and progression of diseases. Newly developed functional genomic approaches, such as serial analysis of gene expression (SAGE) and DNA microarrays have enabled researchers to determine the expression pattern of thousands of genes simultaneously. One attractive feature of SAGE compared to microarrays is its ability to quantify gene expression without prior sequence information or information about genes that are thought to be expressed. SAGE has been successfully applied to the gene expression profiling of a number of human diseases. In this review, we will first discuss SAGE technique and contrast it to microarray. We will then highlight new biological insights that have emerged from its application to the study of human diseases.     

Inhibition of interleukin-1beta-stimulated collagenase and stromelysin expression in human tendon fibroblasts by epigallocatechin gallate ester.

The medicinal benefits of green tea (Camellia sinensis) consumption have been attributed to bioavailable polyphenols, notably epigallocatechin gallate (EGCG). We have assessed the effects of EGCG and its non-esterified counterpart EGC on the expression of the collagenases, matrix metalloproteinases (MMP)-1 and -13, and the stromelysin, MMP-3, in human tendon-derived fibroblasts. Interleukin (IL)-1beta increased MMP-1, -3 and -13 mRNA and output at least 30-fold. EGCG reduced this stimulation, by 20-30% at 2.5 microM and more than 80% at 25 microM, and had a smaller effect on MMP-2 mRNA expression, which was not stimulated by IL-1beta. In all experiments EGCG was at least 10-fold more potent than EGC. EGCG reduced the stimulation of p54 JNK/SAPK phosphorylation by IL-1beta but did not affect p38 MAPK phosphorylation, the degradation of IkappaB or the activating phosphorylation of NFkappaB. We conclude that EGCG reduces the IL-1-stimulated expression of both collagenase and stromelysin mRNA species, an effect which may be mediated by inhibition of the JNK/SAPK pathway. Taken together with previous reports of EGCG effects on the expression and/or activity of gelatinases and aggrecanases, our results underline the importance of extracellular matrix breakdown as a potential target for the actions of green tea polyphenols.     

Growth factor responsiveness of human articular chondrocytes: Distinct profiles in primary chondrocytes,subcultured chondrocytes,and fibroblasts

The objectives of this study were to establish a growth factor response profile for adult human articular chondrocytes, to determine whether this is unique for chondrocytes or influenced by the differentiation status of the cells, and to characterize growth factor interactions. It is shown that transforming growth factor-β (TGF-β) is the most potent mitogen among a variety of factors tested. All three isoforms of TGF-β caused similar dose-dependent increases in chondrocyte proliferation. Other members of the TGF-β family, including bone morphogenetic protein 2B (BMP2B), activin, and inhibin, did not detectably increase chondrocyte proliferation. Platelet-derived growth factor-AA (PDGF-AA), basic fibroblast growth factor (bFGF), and insulin-like growth factor 1 (IGF-1) also stimulated proliferation but were less effective than TGF-β. In contrast to findings with other cell types, the effects of TGF-β on chondrocyte proliferation were not dependent on the endogenous production of PDGF. The cytokines Interleukin 1 (IL-1) and tumor necrosis factor-α (TNF-α) gave no stimulation, but IL-1 inhibited chondrocyte proliferation induced by TGF-β or serum. This response profile was characteristic for primary chondrocytes from human adults and distinct from subcultured (dedifferentiated) chondrocytes or skin fibroblasts. The latter preferentially responded to PDGF, and IL-1 caused greater increases in proliferation than TGF-β. In summary, these results describe growth factor responses that are characteristic for chondrocytes and provide a basis for the analysis of changes in chondrocyte growth proliferation that occur in aging and tissue injury. © 1994 Wiley-Liss, Inc.     

HIV-1 transactivator protein Tat induces proliferation and TGF beta expression in human articular chondrocytes

The human immunodeficiency virus-1 (HIV-1) protein Tat binds to cell surface antigens and can regulate cellular responses. Tat has similar immunosuppressive effects as transforming growth factor-beta (TGF beta) and both inhibit lymphocyte proliferation. TGF beta is expressed by primary human articular chondrocytes and is their most potent growth factor. The present study analyzed the interactions of TGF beta and HIV Tat in the regulation of human articular chondrocytes. Synthetic or recombinant full-length Tat (1-86) induced chondrocyte proliferation and this was of similar magnitude as the response to TGF beta. Tat peptides that did not contain the RGD motif had similar chondrocyte stimulatory activity as full-length Tat. Among a series of Tat peptides, peptide 38-62 which contains the basic domain was the only one active, suggesting that this region is responsible for the effects on chondrocyte proliferation. Full-length Tat and peptide 38-62 synergized with TGF beta and induced proliferative responses that were greater than those obtained with any combination of the known chondrocyte growth factors. Further characterization of the interactions between Tat and TGF beta showed that Tat increased synthesis and TGF beta activity and TGF beta 1 mRNA levels. The stimulatory effects of Tat and peptide 38-62 on chondrocyte proliferation were reduced by neutralizing antibodies to TGF beta and by TGF beta antisense oligonucleotides. These results identify a virally encoded protein and a synthetic peptide derived from it as novel and potent chondrocyte growth stimuli which act at least in part through the induction of TGF beta.     

Recombinant human interleukin-1 stimulates human articular cartilage to undergo resorption and human chondrocytes to produce both tissue- and urokinase-type plasminogen activator

Cytokines capable of stimulating cartilage resorption have frequently been identified as 'interleukin-1 (IL-1)-like' peptides. In this study for the first time we have employed homogeneous recombinant IL-1 alpha and IL-1 beta in an all-human culture system to define the effects of IL-1 on articular cartilage and chondrocytes in culture. Recombinant IL-1 (10-100 U/ml) could stimulate cartilage resorption, although the maximum degree of tissue breakdown rarely reached the levels obtained when cartilage was treated with crude mononuclear-cell conditioned medium or all-trans retinoic acid (1 microM) over a similar time course. Levels of plasminogen activator (PA) activity, a neutral proteinase which may contribute to cartilage destruction in arthritis, increased markedly in the cartilage/chondrocyte culture supernatants and in the chondrocyte cell layers in response to the stimulation of cultures with recombinant IL-1 (1-100 U/ml). Elevated levels of PA activity were detectable after 4-8 h stimulation of the chondrocytes with IL-1 while characterization of the PA activities indicated that both types of PA activity were expressed, viz. urokinase-type PA (u-PA) and tissue-type PA (t-PA). Both IL-1 alpha and IL-1 beta could elicit these responses and their effects were comparable for a given dose. These studies show definitively that pure IL-1, free from contaminating cytokines, is capable of inducing human cartilage resorption and stimulating the expression of two types of PA activity by chondrocytes. In contrast to IL-1, retinoic acid increased the detectable levels of only u-PA in the chondrocyte cell layers. Chondrocyte u-PA may have an important role in cartilage degradative processes since it is one of the few neutral proteinases now known to be increased in activity in retinoid-stimulated cartilage.     

Induction of collagenase mRNA in lapine articular chondrocytes by synovial factors and interleukin-1

The cDNA probe H-9, originally constructed to recognize a portion of the mRNA for lapine synovial collagenase, also hybridized with a RNA of the same size (approximately 2.0 kb) isolated from activated lapine articular chondrocytes. Primary, monolayer cultures of lapine articular chondrocytes did not contain detectable amounts of this RNA, nor did they secrete measurable amounts of collagenase into their culture media. Following exposure to synovial factors, the chondrocytes contained high levels of collagenase mRNA, while their conditioned media had considerable collagenolytic activity. Collagenase mRNA started to appear in chondrocytes 3-5 h after treatment with the synovial material. Maximum levels occurred after 12-24 h. Recombinant human interleukin-1 also induced the appearance of this mRNA. We conclude that chondrocyte collagenase is likely to be the same gene product as synovial collagenase, and that its regulation by lapine articular chondrocytes probably occurs at a pretranslational level.     

Effect of a interleukin-1 like factor (mononuclear cell factor) on proteoglycan synthesis in cultured human articular chondrocytes

A partially purified monocyte factor, with Interleukin-1 properties (MCF/IL-1), enhances the proteoglycan synthesis of human neonatal articular chondrocytes in culture, and changes the repartition of these macromolecules between medium and cell layer. The size of the proteoglycan monomers, the length of the glycosaminoglycan chains and the respective levels of chondroitin-6-sulfate, chondroitin-4-sulfate and non sulfated chondroitin remain unchanged under MCF/IL-1 exposures. The addition of indomethacin reduces the stimulation effect by 60-70% only, suggesting that the MCF/IL-1 action is partially dependent on prostaglandins but seems also related to mechanisms distinct from the cyclooxygenase pathway.     

Stimulation of interleukin-6 release by interleukin-1beta from isolated human adipocytes

The secretion of interleukin-6 (IL-6) is modulated by immune, hormonal and metabolic stimuli in a cell-specific manner. We investigated the effect of cytokines, TNFalpha and IL-1beta, and insulin on IL-6 release from human adipocytes and peripheral blood cells (PBC). Adipocytes released IL-6 constitutively (after 5 h: 5.64 [1.61-15.30]pg ml(-1), after 10 h: 15.95 [2.34-45.59]pg ml(-1), p = 0.007), while PBC secretion did not change significantly over this period. LPS stimulated IL-6 secretion in PBC after 5 h but was without effect on adipocytes. TNFalpha and insulin induced IL-6 production from PBC, but had no effect on adipocytes. IL-1beta, however, induced a substantial increase in IL-6 release in adipocytes and PBC (all p < 0.05). Adipose tissue production of IL-1beta was assessed in vivo by measuring arterio-venous differences across the subcutaneous abdominal adipose bed. Net release of IL-1beta was not observed, suggesting that under basal conditions there is no detectable release of this cytokine into the circulation from this depot. In conclusion (1) PBC demonstrate regulated IL-6 release, while the adipocyte release has a large constitutive component; (2) immune modulators, such as LPS, TNFalpha and IL-1beta, all induce PBC IL-6 release, but only IL-1beta stimulates adipocyte release. Though IL-1beta is not an endocrine signal from adipose tissue, it is an autocrine/paracrine stimulator of IL-6 release from human adipocytes.